This is an application for a K08 award for Dr. Erin Ealba, a general dentist at the University of California, San Francisco. Dr. Ealba is establishing herself as a young investigator in basic science research of craniofacial bone development. This K08 award will provide Dr. Ealba with the support necessary to accomplish the following goals: (1) to become an expert in developmental, skeletal, and craniofacial biology; (2) to focus on faculty skills; and (3) to enhance her manuscript and grant writing abilities. To achieve these goals, Dr. Ealba has assembled a mentoring team comprised of a primary mentor [Dr. Richard Schneider, an authority in the field of craniofacial development], two co-mentors [Dr. Pamela DenBesten, a leading dentist-scientist focused on enamel mineralization, and Dr. Tamara Alliston, an expert in TGF? signaling], and three collaborators [Dr. Mary Nakamura, Dr. Ralph Marcucio, and Dr. Ophir Klein]. Dr. Ealba's long-term goal is to discover novel molecular-based therapies for regulating the length and shape of bone as a means to address the need for non-surgical treatments of craniofacial malformations. The objective of the current study is to build toward this goal by understanding how neural crest mesenchyme (NCM), which forms all the elements in the facial and jaw skeletons, regulates jaw size. To address this issue, we manipulate in vivo the NCM, a highly accessible embryonic population. Specifically, we transplant faster- maturing quail donor NCM into a slower-developing duck host, which creates chimeric quck; and we transplant slower duck donor NCM into the relatively faster quail host, generating chimeric duail. Exploiting the divergent developmental programs of quail and duck provides a unique way to manipulate signaling between NCM and adjacent host tissues, and allows discovery of NCM-dependent processes. Also, all quail cells can be detected via a ubiquitous nuclear marker not present in duck. In published work and in preliminary studies, we observe that NCM autonomously synchronizes and directs osteogenic induction, proliferation, differentiation, matrix deposition, mineralization, and matrix remodeling. How NCM accomplishes such a complex task, and what factors are sufficient to replicate this phenomenon, is unknown. Likely candidates may include members and targets of the Transforming Growth Factor-Beta (TGF2) since they are known to play critical roles during osteogenesis, and their expression is altered in chimeras. Therefore, we hypothesize that by modulating levels of TGF? signaling, NCM directs its own osteogenic program and coordinates the activities of osteoclasts to control jaw length. To test our hypothesis, we propose three complementary and non-interdependent Specific Aims. Specific Aim 1 will determine the extent to which NCM employs TGF2 signaling to control jaw length. Specific Aim 2 will determine the extent to which NCM relies on the actions of osteoclasts to regulate jaw length. Specific Aim 3 will determine the extent to which NCM acts via TGF2-dependent RANKL/OPG signaling to affect osteoclast activity and jaw length. We will employ gain- and loss-of-function techniques to identify molecular mechanisms that endow NCM with the ability to control jaw length. Experiments from this proposal can serve as a proof-of-principle that molecular-based therapies can be devised to treat disorders that affect the length of the jaw. Moreover, identifying mechanisms through which donor NCM transduces its effects on host cells such as osteoclasts has implications for repair and regeneration of bones injured by trauma or diseases like osteoporosis and osteonecrosis. We are hopeful that our research will provide a foundation for biologically based, non-surgical methods to remedy a variety of clinical skeletal conditions.